In August 2022, an outbreak of disease coincident with considerable mortality was occurred in T. fulvidraco, which were cultured in four 5–6 acre earthen ponds in Zhijiang fish farm, Yichang, Hubei province, China. The diseased fish were between 150 and 300 g in body weight, and 15–22 cm in body length. Between 300 and 700 approximated fish deaths were recorded daily for 5 successive days, with mortality rate reached the peak level at around 6th day after first obvious mass mortality. Twenty moribund fish were transported timely to the laboratory in oxygen bags for diagnosis.

The tissues (liver, spleen, head kidney, eye, and brain) were aseptically sampled from moribund fish and streaked directly on brain heart infusion (BHI, HopeBio, China) agar plates with inoculating loop. The agar plates were cultured at 28°C for 48 h and then the bacterial colonies were re-inoculated on BHI agar plates thrice to get the pure culture. The isolate from the above pure culture was inoculated on BHI agar supplemented with 5% defibrinated sheep blood and incubated at 28°C for 48 h to observe colony morphology and hemolytic activity. After that, the morphology of S. iniae from cultured agar and naturally infected fish liver were detected with a Gram Stain Kit (Solarbio, Beijing, China). Images were photographed using 100 × oil immersion objective lens on an optical microscope. The biochemical characteristics of S. iniae were identified using standardized API® 20 strip (BioMerieux S.A., France) according to the guidelines of the manufacturer. The 16S rRNA gene sequencing and phylogenetic tree construction were conducted to identify the bacterial species as reported previously (Xu et al., 2022). Briefly, phylogenetic tree was constructed using the neighbor-joining algorithm in the MEGA X software package following multiple sequence alignments (using CLUSTAL W) of 16S rRNA gene sequence. After that, the 16S rRNA gene sequence of isolated S. iniae was submitted to the National Center for Biotechnology Information (NCBI) database and accession numbers was obtained. In addition, the lactate oxidase (Lox) gene was amplified and visualized on 1% (w/v) agarose gel containing 4S Green for visualization.

Strain 2022SI08 was inoculated in BHI broth at 28°C, kept shaking at 180 rpm for 10 h, and then collected by centrifugation at 10,000 g for 10 min. The total genomic DNA was extracted from isolated bacteria with a TIANamp bacteria DNA Kit (Tiangen, Beijing, China) as per the manufacture’s instruction and subjected to whole genome sequencing using Illumina NovaSeq 6000 sequencing platform (Shanghai Biozeron Biotechnology Co., Ltd) as reported previously (Li S. et al., 2023; Ramadan et al., 2023). Briefly, reads were assembled with SOAP de novo version 2 and the circular genome map was built with Circos software. The coding sequences (CDSs), transfer RNA (tRNA) and ribosomal RNA (rRNA) genes were detected using Glimmer 3.02, GeneMarkS and tRNAscan-SE, respectively. Moreover, the obtained CDSs were annotated using the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, virulence related genes were analyzed by comparison with the virulence factors database (VFDB), and antimicrobial resistance genes were detected through comprehensive antibiotic resistance database (CARD).

The different pH value and NaCl concentration were prepared following previous study with slight modification (Li S. et al., 2023). In brief, the pH values of BHI were adjusted to 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 8.5 with HCl (1 mol/L) or NaOH (1 mol/L) prior to sterilization. Likewise, the concentrations of NaCl in BHI were adjusted to 0.5, 1.0, 1.5, 2.0, 2.5, and 3.0%, respectively, before sterilization. The S. iniae isolate was cultured in BHI broth until the OD600 value reached 1.0. After that, the bacteria were introduced into prepared BHI at a ratio of 1:100 and added 200 μL of each to a 96-well plate. Growth feature of isolated 2022SI08 was monitored for 60 h by reading the OD600 value every 2 h using full-automatic microbial growth curve analyzer (Scientz MGC-200, Ningbo, China).

The S. iniae were conducted for PCR amplification to confirm the presence of genes coding virulence factors, such as C5α peptidase (scpI), polysaccharide deacetylase (pdi), SiM protein A (simA), capsular polysaccharide (cpsB, cpsC, and cpsD), CAMP factor-like (cfi), phosphoglucomutase (pgm), β-haemolysin (tagU, nisF, YkpA, and ydfG), and hyaluronic acid capsule (hasA). The PCR amplifications were performed in reaction volume of 25 μL mixtures containing 1 μL of DNA template (about 100 ng), 2.5 μL of 10 × Taq reaction buffer, 1.0 μL (10 pmol) of each reverse and forward primers, 2.0 μL of dNTP mix (2 mM), 0.5 μL of Taq DNA polymerase (5 U/μL), and 18.0 μL of DNase-free water. Takara thermal cycler TP600 (Takara Bio Inc. Shiga-Ken, Japan) was utilized to provide the following amplification reactions which started with a primary denaturation for 10 min at 95°C, followed by 30 cycles including a secondary denaturation for 30 s at 95°C, annealing at specified temperature (Table 1) for 30 s, extension at 72°C for 45 s, and final extension for 7 min at 72°C. The PCR products were detected by electrophoresis on 1% agarose gel stained with 4S Green. Gels were visualized and photographed using Gel Doc EZ Imager (Bio-Rad, Germany).

The antibiotic susceptibility of isolate 2022SI08 was determined using the disk diffusion method of Bauer (1966). In brief, 100 μL of bacterial suspension was directly swabbed onto Mueller–Hinton agar (Solarbio, Beijing, China) supplemented with 5% sheep blood, and 29 antibiotic disks (Hangzhou Tianhe Microorganism Reagent Co., Ltd., China) were applied on the streaked cultures. The zones of bacterial growth inhibition were measured after 24 h of incubation at 28°C, and antibiotic sensitivity was regarded as susceptible (S), intermediate (I), or resistant (R) according to the Clinical and Laboratory Standards Institute (CLSI) guidelines (CLSI-2016).

The skin, gill, fin, spleen, liver, kidney, intestine, eye, and brain of diseased fish were aseptically sampled and fixed with 10 % neutral buffered paraformaldehyde. Then, the fixed tissues were dehydrated in an alcohol series (50–100%), made transparent in xylene and embedded in paraffin wax. Tissue sections were sliced transversely into 5 μm thicknesses using microtome (Leica Microsystems, Wetzlar, Germany) and stained with hematoxylin and eosin (H&E). The microphotographs were captured under a light microscope (BX51, Olympus, Japan).

The bacterial isolate was cultured on BHI broth for 48 h at 28°C and collected by centrifugation for 5 min at 10,000 g. The bacteria were washed three times and bacterial suspensions were adjusted to 1.2 × 10⁵, 1.2 × 10⁶, 1.2 × 10⁷, 1.2 × 10⁸, and 1.2 × 10⁹ CFU/mL with sterile PBS, respectively. Healthy yellow catfish (n = 400, 20 ± 1 g) were introduced from a commercial hatchery, transported to our laboratory, maintained in 250-L aquaria at 26 ± 0.5°C, and fed with commercial pellets twice daily. Feces and uneaten feed were removed by siphon. During the experiment, the water quality parameters of the aquariums were maintained at Ammonia-N of 0.03 ± 0.01 mg/L, dissolved oxygen at 7.75 ± 0.25 mg/L, and pH at 7.00 ± 0.20, respectively.

For the pathogenicity testing, a total of 180 yellow catfish were randomly divided into six groups (30 fish per group), and then the fish in group 1–5 were intraperitoneally challenged with 100 μL of abovementioned bacterial suspensions. The fish in group 6 was injected with same value of PBS and used as the control. The number of deaths were recorded for 14 consecutive days and the median lethal dosage (LD₅₀) of S. iniae was calculated following previous research (Mabrok et al., 2024).

To analyze the activation of immune response after bacterial infection, a total of 240 yellow catfish were divided randomly into two groups in triplicate. The fish from the experimental group were intraperitoneally challenged with 20 μL of S. iniae suspension at the concentration of 9.53 × 10⁴ CFU/mL, while the fish from control group were injected with an equal volume of sterile PBS. The fish before injection were anesthetized with ethyl 3-aminobenzoate methanesulfonic acid (MS222, Sigma, Beijing, China), and all protocols for experiments were permitted by the Committee of the Ethics on Animal Care and Experiments at Wuhan Polytechnic University (No. WPU202211002).

Blood samples were collected from caudal veins at 12, 24, 48, 72, 120, and 168 h post infection (hpi) and then transferred into 1.5 mL tubes immediately. The samples were incubated at room temperature for 1 h, then centrifuged at 1,200 g for 10 min. Serum samples were obtained from the supernatant and then applied to detect the activities of alkaline phosphatase (AKP), acid phosphatase (ACP), and lysozyme (LZM) with Nanjing Jiancheng Bioengineering Institute kits (Jiangsu, China) as per manufacture’s protocols.

The liver and head kidney of T. fulvidraco were sampled at 12, 24, 48, 72, 120, and 168 hpi, transferred to tubes filled with RNA later solution (TaKaRa, Dalian, China). The transcriptional levels of genes, encoding the cytokines (IL-1β, IL-10, IL-6, TNF-α, and IFN-γ), the chemokines (IL-8, CCL19), the complement factors (C3 and C4), the immuno-globulins (IgD and IgM), the cellular receptors and markers (CD3γ, CD8α, CD83, MHC-2α, and CD28), during the infection were analyzed following the Minimum Information for Publication of Quantitative Real-Time RT-PCR experiments MIQE guidelines (Bustin et al., 2005). The list of primers used in qRT-PCR was described in Table 2. Total RNA was extracted using TRIZOL reagent and the first-strand complementary DNA (cDNA) was synthesized from 1 μg extracted RNA by using cDNA synthesis kit (TaKaRa, Dalian, China) according to the manufacturer’s instruction. The resulting cDNA was then diluted 2-fold with RNAase free water and stored at −80°C before used as template in qPCR assays.

The qPCR was carried out in Thermofisher QuantStudio Real-Time PCR System. The reaction was performed in 20 μL volume containing 1 μL diluted cDNA template, 10 μL 2 × SYBR® SuperMix, 1 μL (10 μM) of each forward and reverse primers and 7 μL of nuclease-free water. The reaction program was 94°C for 5 min, followed by 40 cycles of 94°C for 30 s, 60°C for 15 s, and 72°C for 10 s, then melting from 72 to 95°C. β-actin was utilized as a reference gene for sample normalization, and relative gene expression levels were calculated using the 2^–ΔΔCt method.

All data were expressed as mean ± standard deviation (SD) and analyzed by an un-paired Student’s t-test using Statistical Package for the Social Sciences (SPSS) version 23.0 software for windows (IBM, Armonk, NY, United States). The statistically significant difference was set as p < 0.05.

The diseased fish showed the clinical sign of anorexia, erratic swimming, rotating on the surface, and along with severe mortality (approximate 60%). As shown in Figure 1, the clinical sings of naturally diseased yellow catfish were associated with mass external symptom, including hemorrhages on fin (Figure 1A), mouth (Figure 1B) and eye (Figure 1C), as well as redness and swelling of the anus (Figure 1D). Moreover, some diseased fish showed cerebral oedema and hemorrhaging (Figure 1E). At necropsy, enlarged spleen (Figure 1G) and green excrement (Figure 1F) in the intestine were observed.

The isolate 2022SI08 formed smooth, round, moist, neat-edged and milky white colonies on BHI (Figure 2A, left) and produced β hemolysis on 5% sheep blood agar (Figure 2A, right). Regarding the biochemical tests, the S. iniae 2022SI08 showed acid producers from ribose, glycogen, mannitol, while they were failed to produce acid from lactose, l-arabinose, sorbitol, inulin, and raffinose. The strains produced pyrrolidinyl arylamidase, alkaline phosphatase, and leucine arylamidase; α-galactosidases, β-galactosidases, and acetoin were not produced. Moreover, esculin was hydrolyzed, but arginine dihydrolase and hippurate not (Table 3). The PCR assay resulted in the amplification of approximately 1,500 and 870 bp fragments of 16S rRNA and Lox genes from the isolated 2022SI08, respectively (Figure 2B). The 16S rRNA gene of S. iniae 2022SI08 was deposited in GenBank with an accession No. OQ861164 and displayed highest identity with the 16S rRNA gene of S. iniae strain 2,009,001 (GenBank accession No. MW455461.1) after BLAST alignments. Accordingly, the phylogenetic tree revealed that the isolate 2022SI08 was grouped together with known species of S. iniae (Figure 2C). Moreover, S. iniae from either BHI medium or flesh liver of naturally infected fish showed a positive reaction with blue-purple staining and displayed chain, pair and even single-like cocci morphology, respectively (Figures 2D,E). Based on these data, the isolated 2022SI08 strain from diseased T. fulvidraco was identified phenotypically and molecularly as S. iniae.

The whole genome sequence data of isolate 2022SI08 were deposited at the NCBI BioSample database with accession No. PRJNA907543. The whole genome sequence analysis showed that the total genome of S. iniae 2022SI08 was 1,776,777 bp long, with an average GC content of 37.14% (Figure 3A). A total of 1,909 CDSs, 35 tRNA and three rRNA were identified in the 2022SI08 genome. Besides, 1,490 protein-coding functions were annotated using the KEGG database (Figure 3B). Among these encoded proteins, 61.80% were associated with metabolic functions, 5.17% were related to cellular processes, 10.47% were linked with genetic information processing, 1.68% were related to organismal systems, and 15.03% were associated with environmental information processing functions. Moreover, 5.84% of the genes were related to disease, of which 37 genes were associated with drug resistance and 20 genes were linked to infectious diseases.

The growing characteristics of 2022SI08 isolate on different pH and NaCl values were tested. As shown in Figure 4A, the S. iniae 2022SI08 could grow well at pH 6.5–7.5, whose optimal value was around pH 7. The latent phase of bacterial growth was somewhat prolonged at pH 6.0, and extended significantly when the pH value reached 5.5, whereas the growth was suppressed at pH 8.5. As shown in Figure 4B, the S. iniae 2022SI08 could grow normally at 0.5–1.5% NaCl, whose optimal value was around 1.0% NaCl. Whereas, the latent phase of bacterial growth was extended markedly at 2.0% NaCl and the growth was halted when the NaCl concentration reached 3.0%.

PCR amplification was performed to verify the presence of virulence genes in S. iniae 2022SI08. The results revealed that 15 virulence-related genes (scpI, simA, pdi, sagA, pgm, cfi, cpsB, cpsC, cpsD, tagU, nisF, YkpA, ydfG, and hasA) were positive for isolated S. iniae 2022SI08 (Figure 5A). In this study, a total of 204 virulence genes were screened from the S. iniae 2022SI08 after comparison by whole genome sequence (Supplementary Table S1). These virulence genes were mostly associated with processes of iron uptake, adherence, invasion, and antiphagocytosis (Figure 5B). The yellow catfish injected with 100 μL of 1.2 × 10⁶ to 1.2 × 10⁹ CFU/mL bacteria were rapidly died from the 3rd to 7th day post-injection, accompanied with the symptoms of exophthalmia and pterygiophore hemorrhage, whereas no death was observed in the fish injected with PBS during the experimental challenge. The cumulative mortality of infected fish was displayed in Figure 5C, and the LD₅₀ value of S. iniae 2022SI08 was calculated as 9.53 × 10⁵ CFU/g fish.

The antibiotic resistance profiles of S. iniae, valued in terms of the inhibition zones surrounding each disk, was exhibited in Table 4. Isolate 2022SI08 was resistant (R) to kanamycin, streptomycin, amikacin, gentamycin, cefpimizole, ampicillin, rifampicin, trimethoprim, and sulfisoxazole, intermediate sensitive (I) to minocycline, carbenicillin, meropenem and imipenem, and susceptible (S) to several antibiotics including neomycin, ceftriaxone, ceftazidime, cefepime, doxycycline, tetracycline, piperacillin, oxacillin, enrofloxacin, norfloxacin, ciprofloxacin, levofloxacin, florfenicol, polymyxin B, vancomycin, and colistin.

Besides, the CARD comparison results revealed that 127 antibiotic resistance genes against 25 groups of antibiotics were positive for isolate S. iniae 2022SI08 (Supplementary Table S2), of which coding for macrolide, tetracycline, and fluoroquinolone antibiotics occupy the largest percentage (Supplementary Figure 1).

Histopathological analysis revealed that pathological changes of extensive tissue cell degeneration, necrosis, hemorrhage, and inflammation were observed in the skin, gill, fin, spleen, liver, kidney, intestine, eye, and brain from naturally infected fish. Specifically, the skin tissues of diseased fish showed severe hemorrhage and fibrinoid degeneration with inflammatory cell infiltration (Figure 6A). Large scale of hemorrhage was observed in the fin (Figure 6B). The eyes of diseased fish showed severe hemorrhage, vacuolar degeneration, and necrosis (Figure 6C). The gills of fish infected by S. iniae demonstrated large scale of hemorrhage, severe necrosis, and inflammatory cell infiltration (Figure 6D). Proliferation of melano-macrophage centers, necrosis, fibrinoid degeneration, and lymphocyte infiltration were observed in the spleen (Figure 6E). Severe hemorrhage, necrosis, and inflammatory cell infiltration were observed in both brain and liver (Figures 6F,G). In the kidney, degeneration and tubular necrosis, inflammatory cell infiltration, and proliferation of melano-macrophage centers were detected (Figure 6H). The original structure of cells was disappeared and vacuolated, and more inflammatory cells infiltrated in the interstitium. Necrosis, inflammatory cell infiltration and proliferation of melano-macrophage centers were observed in the intestine (Figure 6I).

The innate immune enzyme activities such as ACP, AKP, and LZM were measured in serum to assess the immune response of yellow catfish infected by S. iniae (Figure 7). The ACP activity in serum was dramatically increased at 24 hpi (p < 0.05), reached the summits at 72 hpi, and then decreased which was remarkably lower than that of control fish at 120 and 168 hpi (p < 0.05). Similarly, the activity of AKP in infected fish was upregulated significantly (p < 0.05) at 12 hpi, reached the maximum at 24 hpi, and then dramatically dropped (p < 0.05) to the value lower than that of control fish at 168 hpi. However, the LZM activity was increased obviously (p < 0.05) in the serum of infected fish than that from the control group.

The transcription profiles of immune associated genes were detected in liver and head-kidney to investigate the immune response of yellow catfish after S. iniae invasion (Figure 8). The transcript profiles of CD83 and CD3γ genes in the liver tissue were increased obviously (p < 0.05) at 48 hpi with S. iniae, reached highest levels at 72 hpi, and then started to decrease but was still remarkably higher (p < 0.05) than that of control fish. The transcript levels of C3, IFN-γ, IL-1β, IL-8, IL-10, C4, TNF-α, IL-6, CD8α, MHC-2α, CD28, IgD, IgM, and CCL19 genes were significantly enhanced (p < 0.05) in the liver at 72 hpi with S. iniae, increased gradually, and finally reached the peak level at 168 hpi. The transcript levels of CCL19, IL-1β, IFN-γ, and C4 in the head kidney were found to be increased apparently (p < 0.05) at 48 h after S. iniae invasion, reached the highest level at 120 hpi, and then decreased but was still obviously greater than that of control group. Whereas, the TNF-α, CD3γ, CD28, IL-6, IL-10, MHC-2α, IgD, CD83, and IgM transcript levels were significantly improved (p < 0.05) in the head kidney of infected fish at 12 hpi in comparison with those in control fish, improved gradually, and then reached summit values at 48, 72, or 120 hpi. The transcript level of C3 gene was increased in the head kidney from 24 to 168 hpi with S. iniae compared to the control group. The CD8α transcripts reached maximum level at 72 hpi, then decreased continuously, and finally returned to the initial level at 168 hpi. The expression of IL-8 was obviously increased (p < 0.05) in the head kidney of infected fish at 72 hpi as compared with that in control fish, improved slightly, and finally reached the highest level at 168 hpi.